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Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA - J. Cell Biol. (2005)

Bottom Line: Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM).This slow release of GLUT4 determined the overall increase of the PM GLUT4.It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

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Traffic of GLUT4 vesicles in adipose cells in the basal and insulin-stimulated states. Time-lapse TIRF images were projected onto a single plane to visualize vesicle movements. (A) Projection image of a cell in the basal state made from a 1-min-long recording (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200412069/DC1). (B) Sequential frames show three vesicles (arrows) taking the same pathway (white line). See Video 2. (C) Projection image of a cell taken 10 min after insulin application. Note that both the number of traces and the average running distance for GLUT4 vesicles are significantly reduced compared with the basal state. Fluorescence intensity is shown in pseudocolor. (D) Quantification of traffic for cells in the basal state and cells 5 and 10 min after insulin application. Data represent mean number of traces detected per 100 μm2/min from at least 20 cells in each state.
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fig2: Traffic of GLUT4 vesicles in adipose cells in the basal and insulin-stimulated states. Time-lapse TIRF images were projected onto a single plane to visualize vesicle movements. (A) Projection image of a cell in the basal state made from a 1-min-long recording (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200412069/DC1). (B) Sequential frames show three vesicles (arrows) taking the same pathway (white line). See Video 2. (C) Projection image of a cell taken 10 min after insulin application. Note that both the number of traces and the average running distance for GLUT4 vesicles are significantly reduced compared with the basal state. Fluorescence intensity is shown in pseudocolor. (D) Quantification of traffic for cells in the basal state and cells 5 and 10 min after insulin application. Data represent mean number of traces detected per 100 μm2/min from at least 20 cells in each state.

Mentions: Time-lapse videos made using TIRFM demonstrated a high basal trafficking of GLUT4 vesicles in the vicinity of the PM (Video 1, available at http://www.jcb.org/cgi/content/full.200412069/DC1). To quantify this traffic, we used a projection algorithm producing distinct traces (Fig. 2 A) for all moving vesicles in the TIRF zone. In basal cells, we detected on average 10.0 ± 1.4 traces/100 μm2/min (22 cells, SD) representing trajectories of moving vesicles. The vesicles often underwent long-range (>10 μm) lateral movements, presumably approaching the PM along the way (as deduced from periodic increases in vesicle fluorescence). Vesicles tended to stop for a period of time (varying from a fraction of a second to 100 s) at dedicated places, and sometimes two or more vesicles stopped at the same location. Ultimately, vesicles exited the TIRF zone laterally or in a direction perpendicular to the coverslip (for a detailed description, see the section Kinetic analysis of GLUT4 recycling in primary adipose cells in the online supplemental material).


Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells.

Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA - J. Cell Biol. (2005)

Traffic of GLUT4 vesicles in adipose cells in the basal and insulin-stimulated states. Time-lapse TIRF images were projected onto a single plane to visualize vesicle movements. (A) Projection image of a cell in the basal state made from a 1-min-long recording (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200412069/DC1). (B) Sequential frames show three vesicles (arrows) taking the same pathway (white line). See Video 2. (C) Projection image of a cell taken 10 min after insulin application. Note that both the number of traces and the average running distance for GLUT4 vesicles are significantly reduced compared with the basal state. Fluorescence intensity is shown in pseudocolor. (D) Quantification of traffic for cells in the basal state and cells 5 and 10 min after insulin application. Data represent mean number of traces detected per 100 μm2/min from at least 20 cells in each state.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171949&req=5

fig2: Traffic of GLUT4 vesicles in adipose cells in the basal and insulin-stimulated states. Time-lapse TIRF images were projected onto a single plane to visualize vesicle movements. (A) Projection image of a cell in the basal state made from a 1-min-long recording (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200412069/DC1). (B) Sequential frames show three vesicles (arrows) taking the same pathway (white line). See Video 2. (C) Projection image of a cell taken 10 min after insulin application. Note that both the number of traces and the average running distance for GLUT4 vesicles are significantly reduced compared with the basal state. Fluorescence intensity is shown in pseudocolor. (D) Quantification of traffic for cells in the basal state and cells 5 and 10 min after insulin application. Data represent mean number of traces detected per 100 μm2/min from at least 20 cells in each state.
Mentions: Time-lapse videos made using TIRFM demonstrated a high basal trafficking of GLUT4 vesicles in the vicinity of the PM (Video 1, available at http://www.jcb.org/cgi/content/full.200412069/DC1). To quantify this traffic, we used a projection algorithm producing distinct traces (Fig. 2 A) for all moving vesicles in the TIRF zone. In basal cells, we detected on average 10.0 ± 1.4 traces/100 μm2/min (22 cells, SD) representing trajectories of moving vesicles. The vesicles often underwent long-range (>10 μm) lateral movements, presumably approaching the PM along the way (as deduced from periodic increases in vesicle fluorescence). Vesicles tended to stop for a period of time (varying from a fraction of a second to 100 s) at dedicated places, and sometimes two or more vesicles stopped at the same location. Ultimately, vesicles exited the TIRF zone laterally or in a direction perpendicular to the coverslip (for a detailed description, see the section Kinetic analysis of GLUT4 recycling in primary adipose cells in the online supplemental material).

Bottom Line: Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM).This slow release of GLUT4 determined the overall increase of the PM GLUT4.It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Glucose transport in adipose cells is regulated by changing the distribution of glucose transporter 4 (GLUT4) between the cell interior and the plasma membrane (PM). Insulin shifts this distribution by augmenting the rate of exocytosis of specialized GLUT4 vesicles. We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of this GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted this traffic by tightly tethering vesicles to the PM where they formed clusters and slowly fused to the PM. This slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.

Show MeSH
Related in: MedlinePlus